MicroRNA profiling predicts positive nodal status in papillary thyroid carcinoma in the preoperative setting.

BACKGROUND: The molecular characterization of thyroid nodules in cytological samples has so far been focused on discriminating between benign and malignant forms in a purely diagnostic setting. The evidence on the impact of molecular biomarkers to determine the risk of aggressiveness in cytologically "neoplastic" lesions is limited to genomic alterations (such as BRAF and TERT mutations). The aim of our study was to assess the preoperative role of microRNAs (miRNAs) in predicting the nodal status of patients with papillary thyroid cancer. METHODS: A pilot series of histological samples of papillary thyroid carcinoma with (6 cases) or without (6 cases) lymph node metastases, matched for other major clinical and pathological features, was analyzed for global miRNA expression in a screening phase. A set of miRNAs was then validated in a series of 63 consecutive cytological samples of papillary carcinomas: 48 pN-negative and 15 pN-positive at histology. RESULTS: Unsupervised cluster analysis segregated surgical pN-negative and pN-positive samples, except for 1 case. The 45 differentially expressed miRNAs in pN-positive versus pN-negative cases were predicted to regulate a wide range of cellular pathways, enriched for Wnt, gonadotropin-releasing hormone receptor, and cerulein/cholecystokinin receptor signaling. In agreement with their profiles in surgical samples, 4 miRNAs of the 10 selected for validation (miR-154-3p, miR-299-5p, miR-376a-3p, and miR-302E) had a significant differential expression in cytological samples of papillary carcinoma with lymph node metastases and predicted the positive nodal status with a relatively good performance. CONCLUSIONS: MiRNA profiling is a potential promising strategy to define papillary carcinoma aggressiveness in the preoperative setting.

Protective effects of baicalin on caerulein-induced AR42J pancreatic acinar cells by attenuating oxidative stress through miR-136-5p downregulation.

Baicalin, the main active component of Scutellaria baicalensis, has antioxidant and anti-apoptotic effects and is used to treat acute pancreatitis; however, its specific mechanism is unclear. This study aims to determine the protective effect and underlying mechanism of baicalin on AR42J pancreatic acinar cell injury. AR42J acinar cells (caerulein, 10 nmol/L) were induced in vitro to establish a cell model for acute pancreatitis. Cell relative survival was measured by thiazolyl blue tetrazolium bromide, and cell apoptosis and death were examined by flow cytometry. The expression levels of superoxide dismutase1 (SOD1), Bax, survivin, Bcl-2, caspase-3, and caspase-7 proteins were analyzed by Western blot, and those of SOD1 mRNA and miR-136-5p were determined by RT-PCR. The activities of GSH, SOD1, ROS, and MDA were also investigated. Compared with those of the caerulein group, the relative survival rate and activity of AR42J pancreatic acinar cells with different baicalin concentrations were significantly increased (p < 0.05), and the supernatant amylase level was markedly decreased (p < 0.05). In addition, the ROS and MDA activities and mir-136-5p expression were significantly decreased, and the GSH activities and SOD1 gene and protein expression levels were markedly increased (p < 0.05). These results suggest that baicalin reduced the caerulein-induced death of AR42J acinar cells and alleviated the caerulein-induced injury in pancreatic acinar cells by inhibiting oxidative stress. The mechanism may be related to the decreased expression of Mir-136-5p and the increased expression of SOD1 gene and protein.

Quantitative Imaging of Ca2+ by 3D-FLIM in Live Tissues.

The calcium concentration within living cells is highly dynamic and, for many cell types, a reliable indicator of the functional state of the cells-both of isolated cells, but even, more important, of cells in tissue. In order to dynamically quantify intracellular calcium levels, various genetically encoded calcium sensors have been developed-the best of which are those based on Förster resonant energy transfer (FRET). Here we present a fluorescence lifetime imaging (FLIM) method to measure FRET in such a calcium sensor (TN L15) in neurons of hippocampal slices and of the brain stem of anesthetized mice. The method gives the unique opportunity to determine absolute neuronal calcium concentrations in the living organism.

The oncogenic microRNA miR-21 promotes regulated necrosis in mice.

MicroRNAs (miRNAs) regulate apoptosis, yet their role in regulated necrosis remains unknown. miR-21 is overexpressed in nearly all human cancer types and its role as an oncogene is suggested to largely depend on its anti-apoptotic action. Here we show that miR-21 is overexpressed in a murine model of acute pancreatitis, a pathologic condition involving RIP3-dependent regulated necrosis (necroptosis). Therefore, we investigate the role of miR-21 in acute pancreatitis injury and necroptosis. miR-21 deficiency protects against caerulein- or L-arginine-induced acute pancreatitis in mice. miR-21 inhibition using locked-nucleic-acid-modified oligonucleotide effectively reduces pancreatitis severity. miR-21 deletion is also protective in tumour necrosis factor-induced systemic inflammatory response syndrome. These data suggest that miRNAs are critical participants in necroptosis and miR-21 enhances cellular necrosis by negatively regulating tumour suppressor genes associated with the death-receptor-mediated intrinsic apoptosis pathway, and could be a therapeutic target for preventing pathologic necrosis.

Ligand-induced rearrangements of the GABA(B) receptor revealed by fluorescence resonance energy transfer.

The gamma-aminobutyric acid type B receptor (GABA(B)R), one of the family C G-protein-coupled receptor members, exists as a heterodimer comprised of subunits GB1 and GB2. To clarify the ligand-induced activation mechanism of the GABA(B)R, each subunit was fused with either Cerulean or enhanced yellow fluorescent protein at its intracellular loop, and fluorescence resonance energy transfer (FRET) changes upon agonist application were monitored. As a result, FRET decreases were observed between GB1a loop 2 and GB2 loop 2 and between GB1a loop 2 and GB2 loop 1, suggesting the dissociation of intracellular domains during the receptor activation. Both intersubunit FRET pairs were expected to faithfully capture the activation of the original receptor as their pharmacological properties were highly similar to that of the wild-type receptor. However, the intrasubunit data suggest that the receptor activation does not involve major structural changes within the transmembrane domain of each subunit. By combining the results obtained from two different levels, it was concluded that the GABA(B)R activation by agonist is associated with an asymmetrical intersubunit rearrangement of GB1a and GB2 on the membrane. This type of activation mode, an intersubunit rearrangement without apparent intrahelical structural changes, appears commonly shared by the GABA(B)R and the metabotropic glutamate receptor 1alpha, another family C G-protein-coupled receptor previously studied by our group. Nevertheless, the directions of intracellular domain movements and its asymmetry observed here highlight the qualitative difference between the two receptors.

Identical skin toxins by convergent molecular adaptation in frogs.

The Tree of Life is rife with adaptive convergences at all scales and biological levels of complexity. However, natural selection is not likely to result in the independent evolution of identical gene products. Here we report such a striking example of evolutionary convergence in the toxic skin secretions of two distantly related frog lineages. Caeruleins are important decapeptides in pharmacological and clinical research [1] and are commonly believed to represent a single evolutionary class of peptides [2-4]. Instead, our phylogenetic analyses combining transcriptome and genome data reveal that independently evolved precursor genes encode identical caeruleins in Xenopus and Litoria frogs. The former arose by duplication from the cholecystokinin (cck) gene, whereas the latter was derived from the gastrin gene. These hormone genes that are involved in many physiological processes diverged early in vertebrate evolution, after a segmental duplication during the Cambrian period. Besides implicating convergent mutations of the peptide-encoding sequence, recurrent caerulein origins entail parallel shifts of expression from the gut-brain axis to skin secretory glands. These results highlight extreme structural convergence in anciently diverged genes as an evolutionary mechanism through which recurrent adaptation is attained across large phylogenetic distances.

Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer.

Protein labeling with green fluorescent protein derivatives has become an invaluable tool in cell biology. Protein quantification, however, is difficult when cells express constructs with overlapping fluorescent emissions. Under these conditions, signal separation using emission filters is inherently inefficient. Spectral imaging solves this problem by recording emission spectra directly. Unfortunately, linear unmixing, the algorithm used for quantifying individual fluorophores from emission spectra, fails when resonance energy transfer (RET) is present. We therefore sought to develop an unmixing algorithm that incorporates RET. An equation for spectral emission incorporating RET was derived and an assay based on this formalism, spectral RET (sRET), was developed. Standards with defined RET efficiencies and with known Cerulean/Venus ratios were constructed and used to test sRET. We demonstrate that sRET analysis is a comprehensive, photon-efficient method for imaging RET efficiencies and accurately determines donor and acceptor concentrations in living cells.

Structure of two cDNAs encoding cholecystokinin precursors from the brain of Xenopus laevis.

The skin secretions of many frogs, including Xenopus laevis, contain caerulein, a peptide related to mammalian cholecystokinin. We have screened a cDNA library prepared from the brain of this frog using a cloned cDNA encoding one of the caerulein precursors as a probe. Two clones were isolated which contained inserts encoding cholecystokinin precursors. It was found that the predicted precursor polypeptides resembled their mammalian counterparts rather than the caerulein precursors from the same species. The corresponding mRNAs of different size encoding the Xenopus cholecystokinin precursors are expressed in brain and in the gastrointestinal tract, but not in skin. The smaller mRNA was also detected in lung. These data demonstrate that a polypeptide homologous to mammalian cholecystokinin precursors was present early in the evolution of vertebrates. The possible evolution of the genes encoding the more complex caerulein precursors is discussed.

The genes for the frog skin peptides GLa, xenopsin, levitide and caerulein contain a homologous export exon encoding a signal sequence and part of an amphiphilic peptide.

From genomic libraries of Xenopus laevis, parts of the genes coding for the precursors of the skin peptides GLa (peptide with amino-terminal glycine and carboxy-terminal leucinamide), xenopsin and levitide have been isolated and sequenced. The gene for prepropeptide GLa comprises four exons, separated by relatively small introns. The gene for preproxenopsin is composed of five exons, of which all but the last one have been analyzed. This is a large gene encompassing at least 25,000 base pairs. In addition, two exons of the gene for preprolevitide have been isolated. A comparison of these genes reveals the presence of a homologous exon. This exon contains 161 bp, starts one base pair prior to the initiation codon and encodes a signal peptide and part of a pro region with processing sites. In addition, the two genes for preprocaerulein analyzed previously [Vlasak et al. (1987) Eur. J. Biochem. 169, 53-58] also contain a similar exon. This demonstrates the existence of a homologous export exon in genes encoding the precursors of different skin peptides.

Relationship of promagainin to three other prohormones from the skin of Xenopus laevis: a different perspective.

We observed a striking sequence similarity between precursors for promagainin and procaerulein type I (excluding the caerulein peptide region). Additional comparisons of the promagainin precursor with those of other procaeruleins, proxenopsin, and peptide-Gly-Leu-amide revealed that all possess one or more copies of a structurally similar spacer module, from which an amphiphilic spacer peptide is cleaved. Promagainin yields the magainins, spacer peptides with antimicrobial activity; we suggest other spacer peptides may have similar activity. We propose that the genes for the four kinds of hormones were derived from a common ancestral gene through gene and exon duplications and that the procaerulein and proxenopsin genes are mosaic genes in which the original 3'-ends were replaced by exon shuffling.

Interactive molecular biology computing.

It is clear that the selection of the best possible algorithm for a computer program is essential for the creation of a useful tool. After this first step is taken, however, the usefulness of such a program may be greatly enhanced or impeded by the way it is implemented. We illustrate this point by describing our implementation of the well known FASTP/FASTIN algorithm in an interactive software environment.

Conserved exon-intron organization in two different caerulein precursor genes of Xenopus laevis. Additional detection of an exon potentially coding for a new peptide.

From a genomic library of Xenopus laevis, two genes coding for different preprocaeruleins have been isolated and sequenced. These correspond to the type I and type III precursors analyzed previously at the cDNA level [Richter, K., Egger, R. and Kreil, G. (1986) J. Biol. Chem. 261, 3676-3680]. The type III gene comprises eight exons; the type I apparently contains eight exons as well, of which six have been sequenced. The genetic information for the dekapeptide caerulein is present on small exons of 45 base pairs. The two genes are highly homologous in their 5'-flanking region, the exon/intron boundaries, and long stretches of intron sequences. A possible scheme for the evolution of this small family of genes through exon and gene duplications is presented. In the type I gene, in place of one of the caerulein exons, a potential exon with conserved splice sites was discovered. If expressed in some frog cells, this exon would code for a new peptide 60% homologous to caerulein.

Cerulein mRNA and peptide alpha-amidation activity in the skin of Xenopus laevis: stimulation by norepinephrine.

The skin of Xenopus laevis, because of its high concentrations of amidated peptides, is an excellent model for the study of neuropeptide biosynthesis. In this paper we determined levels of the mRNAs which encode cerulin and characterized the enzymatic activity which likely produces the carboxyl terminal alpha-amide moiety of cerulein. This enzyme is stimulated by the addition of CuSO4 and ascorbate and requires the presence of molecular oxygen and is thus similar to the peptidyl-glycine alpha-amidating monooxygenase (PAM) described in mammals. Hybridization analysis showed that three cerulein mRNAs of different molecular sizes are present in varying proportions in the skin of individual frogs. Injection of norepinephrine into the dorsal lymph sac of Xenopus caused immediate secretion and depletion from skin of PAM activity but did not cause a comparable immediate decrease in skin levels of cerulein mRNA or total RNA. By 2 weeks after norepinephrine injection, cerulein mRNA levels increased to fourfold over control levels. By 4 weeks, skin PAM activity was almost restored to control levels.

Skin peptides in Xenopus laevis: morphological requirements for precursor processing in developing and regenerating granular skin glands.

The biosynthesis of the peptides caerulein and PGLa in granular skin glands of Xenopus laevis proceeds through a pathway that involves discrete morphological rearrangements of the entire secretory compartment. Immunocytochemical localization of these peptides during gland development indicates that biosynthetic precursors are synthesized in intact secretory cells, whereas posttranslational processing requires morphological reorganization to a vacuolated stage. The bulk of the processed secretory material is then stored in vacuolae-derived storage granules. In the mature gland, storage granules are still formed at a low level. However, in this case processing takes place in a distinct cytoplasmic structure, the multicored body, which we suggest to be functionally equivalent to vacuolae. When granular glands regenerate after having lost all their storage granules upon strong stimuli, another morphological pathway is used. 2 wk after gland depletion, secretory cells become arranged in a monolayer that covers the luminal surface of the gland. Storage granules are formed continuously within these intact secretory cells. Here, precursor processing does not require a vacuolated stage as in newly generated glands but occurs in multicored bodies. Most storage granules seem to be formed in the third week of regeneration. The high biosynthetic activity is also reflected by the high activity of the putative processing enzyme dipeptidyl aminopeptidase during this period of regeneration.

Sequence of preprocaerulein cDNAs cloned from skin of Xenopus laevis. A small family of precursors containing one, three, or four copies of the final product.

From skin of Xenopus laevis, cDNA libraries were constructed and clones coding for the precursors of caerulein were isolated and sequenced. Using restriction endonuclease digestions, three different types of preprocaerulein cDNAs could be discerned. These were termed types I, III, and IV in accordance with the number of caerulein copies present in the sequence, the type III being the most abundant one. An incomplete copy of a fourth variant, termed type I', was also found. Besides deletions/insertions encompassing one or two caerulein sequences, these types also differ from each other by several point mutations. In the homologous precursor polypeptides deduced from the nucleotide sequence of these cloned cDNAs, the caerulein copies are flanked by complex processing sequences. These are Arg-Arg-Phe-Ala-Asp-Gly or Arg-Arg-Asp-Gly at the amino-terminal side and Gly-Arg-Arg at the carboxyl end. Between caerulein copies, highly homologous segments are present both at the polypeptide and cDNA level. This homology is evident both within a given precursor, where up to three such segments are present, and between the different types of precursors. We conclude that preprocaerulein cDNAs in the skin of X. laevis represent a small family, at least part of which is derived from different genes rather than being formed by alternative splicing of pre-mRNAs.

Complete nucleotide sequence of mRNA for caerulein precursor from Xenopus skin: the mRNA contains an unusual repetitive structure.

The complete nucleotide sequence of mRNA for caerulein precursor in the skin of Xenopus laevis was determined. The sequence was composed of 705 bp of coding region, accounting for 234 amino acids, 58 bp of 5'-untranslated region and 158 bp of 3'-untranslated region containing two putative poly(A) signals. It coded for four caerulein peptides interspersed with three 147 bp segments (intercaerulein segment; ICS). Analyses of several caerulein encoding cDNAs revealed some interesting features of caerulein mRNA species, which were highly heterogeneous and consisted of a repetition of two fundamental RNA sequences, a 45-nucleotide caerulein fragment and a 147-nucleotide ICS. The result of Northern blotting indicated that caerulein mRNA was only present in frog skin, not in stomach, upper intestine or liver. It appears that caerulein has different physiological function(s) from mammalian gastrin and cholecystokinin-pancreozymin (CCK). The relationship of caerulein to mammalian gastrointestinal hormones is discussed.

Biosynthesis of peptides in the skin of Xenopus laevis: isolation of novel peptides predicted from the sequence of cloned cDNAs.

From skin of Xenopus laevis, a few peptides have been isolated which are identical or homologous to gastrointestinal hormones and/or neurotransmitters of mammalian origin. We have studied the biosynthesis of these peptides using recombinant DNA techniques. From cDNA librariers constructed from skin mRNA, clones with inserts coding for the precursors of caerulein, thyrotropin releasing hormone and a new peptide termed PGLa have been isolated and sequenced. In the case of caerulein, a small family of precursors containing one, three or four copies of the end product have been detected. The caerulein sequences are separated by homologous sequences which potentially could give rise to additional constituents of skin secretion. Three such peptides have been detected which are presumably liberated from caerulein precursors by cleavage at single arginine residues.

An unusual repetitive structure of caerulein mRNA from the skin of Xenopus laevis.

The nucleotide sequence of a 784-bp segment of cloned caerulein mRNA obtained from the skin of Xenopus laevis was determined. It codes for five heterogeneous procaerulein peptides interspersed with three 147-bp intercaerulein segments (ICS). The ICSs contain six inverted repeats and five eukaryotic enhancer-like sequences. Evidence for the presence of multiple forms of caerulein mRNA is presented.

Biosynthesis of caerulein in the skin of Xenopus laevis: partial sequences of precursors as deduced from cDNA clones.

The decapeptide caerulein represents one of the main constituents of the skin secretion of Xenopus laevis. Total mRNA was isolated from skin, transcribed into cDNA and inserted via GC-tailing into the plasmid pUC8. Among the transformants, 300 clones were selected at random and screened with a cDNA primed with the synthetic deoxynucleotide d(AGTCCATCCA), which is complementary to the mRNA region coding for the fragment Trp-Met-Asp-Phe of cerulein. Of nine strongly hybridizing clones, three were sequenced and these were found to contain inserts with very similar nucleotide sequences. The cloned cDNAs code for parts of two different caerulein precursors. These contain one or two copies of caerulein and five additional amino acids located between pairs of arginine residues. The extra glycine at the carboxy terminus is considered to serve as the signal for amidation, while the tetrapeptide Phe-Ala-Asp-Gly, linked to the amino end of caerulein in these precursors, must be cleaved by an unusual processing mechanism.